Unequal SCE is a rare event in homologous recombination between duplicated neo gene fragments in CHO cells.
The frequency of sister-chromatid exchange (SCE) was studied in Chinese hamster ovary (CHO) cell lines with stable insertions of the vector pIII-14gpt which contains 2 truncated neomycin resistance (neo) gene fragments. Recombination between regions of homology in the 2 fragments can restore a functional neo gene and make the cell resistant to the antibiotic G418, a neomycin analogue. Unequal SCE would be one of several possible mechanisms for this event. The observed spontaneous rate of formation of G418-resistant subclones was approximately 6.4 x 10(-6) per cell per generation, as compared to the estimated spontaneous frequency of 3 SCE per cell per generation. Given this SCE frequency, the probability of an SCE occurring in a target site of about 1600 bp (the distance separating the homologous regions in the neo fragments) would be about 8 x 10(-7) per cell per generation, or approximately one tenth of the estimated rate of recombination. Treatment of the cells with methyl methanesulfonate (MMS, 50 x 10(-6) M) induced about 80-90 SCE per cell, corresponding to a probability of 2 x 10(-5) SCE per 1600-bp target per cell. In the same cell culture, MMS treatment induced 4-8 x 10(-4) recombination events per cell giving rise to G418 resistance. Cells treated with HN2 (up to 4 x 10(-6) M) showed a significant increase in SCEs, but no change in the frequency of G418-resistant revertants. These results suggest that the 2 pathways leading to SCE and recombination respectively are uncoupled, and only a small fraction of the recombination events, if any, are due to unequal SCE in this system.
Hellgren
D
D
Karolinska Institutet, Department of Clinical Genetics, Karolinska Hospital, Stockholm, Sweden.
Sahlén
S
S
Lambert
B
B
D000123:Acetyltransferases / Q000235:genetics; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004274:DNA, Recombinant; D004351:Drug Resistance; D005839:Gentamicins / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D011995:Recombination, Genetic / Q000187:drug effects; D012854:Sister Chromatid Exchange / Q000187:drug effects
D004274:DNA, Recombinant; D005839:Gentamicins; C010680:antibiotic G 418; D008741:Methyl Methanesulfonate; D000123:Acetyltransferases; C019681:aminoglycoside acetyltransferase